Interplay in galectin expression predicts patient outcomes in a spatially restricted manner in PDAC.

BACKGROUND: Galectins (Gal's) are a family of carbohydrate-binding proteins that are known to support the tumour microenvironment through their immunosuppressive activity and ability to promote metastasis. As such they are attractive therapeutic targets, but little is known about the cellular expression pattern of galectins within the tumour and its neighbouring stromal microenvironment. Here we investigated the cellular expression pattern of Gals within pancreatic ductal adenocarcinoma (PDAC). METHODS: Galectin gene and protein expression were analysed by scRNAseq (n=4) and immunofluorescence imaging (n=19) in fibroblasts and epithelial cells of pancreatic biopsies from PDAC patients. Galectin surface expression was also assessed on tumour adjacent normal fibroblasts and cancer associated primary fibroblasts from PDAC biopsies using flow cytometry. RESULTS: scRNAseq revealed higher Gal-1 expression in fibroblasts and higher Gal-3 and -4 expression in epithelial cells. Both podoplanin (PDPN+, stromal/fibroblast) cells and EpCAM+ epithelial cells expressed Gal-1 protein, with highest expression seen in the stromal compartment. By contrast, significantly more Gal-3 and -4 protein was expressed in ductal cells expressing either EpCAM or PDPN, when compared to the stroma. Ductal Gal-4 cellular expression negatively correlated with ductal Gal-1, but not Gal-3 expression. Higher ductal cellular expression of Gal-1 correlated with smaller tumour size and better patient survival. CONCLUSIONS: In summary, the intricate interplay and cell-specific expression patterns of galectins within the PDAC tissue, particularly the inverse correlation between Gal-1 and Gal-4 in ducts and its significant association with patient survival, highlights the complex molecular landscape underlying PDAC and provides valuable insights for future therapeutic interventions.

Spatial determination and prognostic impact of the fibroblast transcriptome in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma has a poor clinical outcome and responses to immunotherapy are suboptimal. Stromal fibroblasts are a dominant but heterogenous population within the tumor microenvironment and therapeutic targeting of stromal subsets may have therapeutic utility. Here, we combine spatial transcriptomics and scRNA-Seq datasets to define the transcriptome of tumor-proximal and tumor-distal cancer-associated fibroblasts (CAFs) and link this to clinical outcome. Tumor-proximal fibroblasts comprise large populations of myofibroblasts, strongly expressed podoplanin, and were enriched for Wnt ligand signaling. In contrast, inflammatory CAFs were dominant within tumor-distal subsets and expressed complement components and the Wnt-inhibitor SFRP2. Poor clinical outcome was correlated with elevated HIF-1α and podoplanin expression whilst expression of inflammatory and complement genes was predictive of extended survival. These findings demonstrate the extreme transcriptional heterogeneity of CAFs and its determination by apposition to tumor. Selective targeting of tumor-proximal subsets, potentially combined with HIF-1α inhibition and immune stimulation, may offer a multi-modal therapeutic approach for this disease.

Pancreatic cancer is one of the deadliest and most difficult cancers to treat. It responds poorly to immunotherapy for instance, despite this approach often succeeding in enlisting immune cells to fight tumours in other organs. This may be due, in part, to a type of cell called fibroblasts. Not only do these wrap pancreatic tumours in a dense, protective layer, they also foster complex relationships with the cancerous cells: some fibroblasts may fuel tumour growth, while other may help to contain its spread. These different roles may be linked to spatial location, with fibroblasts adopting different profiles depending on their proximity with cancer calls. For example, certain fibroblasts close to the tumour resemble the myofibroblasts present in healing wounds, while those at the periphery show signs of being involved in inflammation. Being able to specifically eliminate pro-cancer fibroblasts requires a better understanding of the factors that shape the role of these cells, and how to identify them. To examine this problem, Croft et al. relied on tumour samples obtained from pancreatic cancer patients. They mapped out the location of individual fibroblasts in the vicinity of the tumour and analysed their gene activity. These experiments helped to reveal the characteristics of different populations of fibroblasts. For example, they showed that the myofibroblast-like cells closest to the tumour exhibited signs of oxygen deprivation; they also produced podoplanin, a protein known to promote cancer progression. In contrast, cells further from the cancer produced more immune-related proteins. Combining these data with information obtained from patients' clinical records, Croft et al. found that samples from individuals with worse survival outcomes often featured higher levels of podoplanin and hypoxia. Inflammatory markers, however, were more likely to be present in individuals with good outcomes. Overall, these findings could help to develop ways to selectively target fibroblasts that support the growth of pancreatic cancer. Weakening these cells could in turn make the tumour accessible to immune cells, and more vulnerable to immunotherapies.

Tissue-Resident Memory T Cells in Pancreatic Ductal Adenocarcinoma Coexpress PD-1 and TIGIT and Functional Inhibition Is Reversible by Dual Antibody Blockade.

Pancreatic ductal adenocarcinoma (PDAC) has a poor clinical outlook. Responses to immune checkpoint blockade are suboptimal and a much more detailed understanding of the tumor immune microenvironment is needed if this situation is to be improved. Here, we characterized tumor-infiltrating T-cell populations in patients with PDAC using cytometry by time of flight (CyTOF) and single-cell RNA sequencing. T cells were the predominant immune cell subset observed within tumors. Over 30% of CD4+ T cells expressed a CCR6+CD161+ Th17 phenotype and 17% displayed an activated regulatory T-cell profile. Large populations of CD8+ tissue-resident memory (TRM) T cells were also present and expressed high levels of programmed cell death protein 1 (PD-1) and TIGIT. A population of putative tumor-reactive CD103+CD39+ T cells was also observed within the CD8+ tumor-infiltrating lymphocytes population. The expression of PD-1 ligands was limited largely to hemopoietic cells whilst TIGIT ligands were expressed widely within the tumor microenvironment. Programmed death-ligand 1 and CD155 were expressed within the T-cell area of ectopic lymphoid structures and colocalized with PD-1+TIGIT+ CD8+ T cells. Combinatorial anti-PD-1 and TIGIT blockade enhanced IFNγ secretion and proliferation of T cells in the presence of PD-1 and TIGIT ligands. As such, we showed that the PDAC microenvironment is characterized by the presence of substantial populations of TRM cells with an exhausted PD-1+TIGIT+ phenotype where dual checkpoint receptor blockade represents a promising avenue for future immunotherapy.

NK cells in pancreatic cancer demonstrate impaired cytotoxicity and a regulatory IL-10 phenotype.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common tumor subtypes and remains associated with very poor survival. T cell infiltration into tumor tissue is associated with improved clinical outcome but little is known regarding the potential role of NK cells in disease control. Here we analyze the phenotype and function of NK cells in the blood and tumor tissue from patients with PDAC. Peripheral NK cells are present in normal numbers but display a CD16hiCD57hi phenotype with marked downregulation of NKG2D. Importantly, these cells demonstrate reduced cytotoxic activity and low levels of IFN-γ expression but instead produce high levels of intracellular IL-10, an immunoregulatory cytokine found at increased levels in the blood of PDAC patients. In contrast, NK cells are largely excluded from tumor tissue where they display strong downregulation of both CD16 and CD57, a phenotype that was recapitulated in primary NK cells following co-culture with PDAC organoids. Moreover, expression of activatory proteins, including DNAM-1 and NKP30, was markedly suppressed and the DNAM-1 ligand PVR was strongly expressed on tumor cells. As such, in situ and peripheral NK cells display differential features in patients with PDAC and indicate local and systemic mechanisms by which the tumor can evade immune control. These findings offer a number of potential options for NK-based immunotherapy in the management of patients with PDAC.